Methods of increasing recombinant host protein production in the pharmaceutical industry and in the laboratory are highly desirable in many ways, including cost savings, times savings, and manufacturing capacity. Treatment with sodium butyrate has been one means of increasing protein production in cell culture in commercial biopharmaceutical processes. However, the derived benefit of increased protein yields is sometimes offset by the toxic side effects of sodium butyrate.
Sodium butyrate is a short chain fatty acid that inhibits the histone deacetylase (HDAC) enzyme responsible for the maintenance of chromatin structure in the nucleus of cells (Davie, J. Nutrition 133: 2485S-2493S, 2003). The loss of activity results in an alteration in transcriptional regulation of genes through the normal acetylation and deacetylation process of histones (Prasad et al., In Vitro 12: 125-132, 1976). The change in transcriptional regulation has been shown to increase the specific productivity of cell lines producing recombinant proteins in vitro. For example, sodium butyrate has been shown to increase the synthesis of secreted recombinant follicle stimulating hormone (FSH), tissue plasminogen activator (tPA), erythropoietin (EPO), and thrombopoietin (TPO) in Chinese hamster ovary (CHO) cells (Sung et al., J. Biotechnology 112: 323-335, 2004; Hendrick et al. Cytotechnology 36: 71-83, 2001; Chung et al., J. Microbiol. Biotechnol. 11, 1087-1092, 2001; Chotigeat et al., Cytotechnology 15: 217-221, 1994; Chang et al., Free Radical Research 30: 85-91, 1999). The precise mechanisms responsible for these increases are uncertain. Sodium butyrate treatment has been shown to transiently increase mRNA levels for recombinant protein, resulting in increases in resulting protein biosynthesis (Yuan et al., J. Biol. Chem. 260: 3778-3783, 1985).
Changes in gene expression caused by sodium butyrate have been studied previously in cell lines involved in colon cancer research. The studies demonstrated that sodium butyrate alters the expression of multiple genes involved in cell cycle progression, differentiation, cytokine signaling, and apoptosis. However, such studies were limited to relatively a small subset of genes and did not determine whether genes were involved in protein biosynthesis. (Joseph et al., Oncogene 23: 6304-6315, 2004; Tabuchi et al., Biochem. Biophys. Research Comm. 293: 1287-1294, 2002; Iacomino et al., Biochem. Biophys. Research Comm. 285: 1280-1289, 2001; Mariadason et al., Cancer Res. 60: 4561-4572, 2000; Della Ragione et al., FEBS Letters 499, 199-204, 2001).
Alpha 1,2 mannosidase I enzyme (MAN1C1) is an enzyme involved in glycoprotein N-linked oligosaccharide processing that has been described in Tremblay et al. (Glycobiology 8: 585-595, 1998) and Gonzalez et al. (J. Biol. Chem. 274: 21375-21386, 1999). The enzyme catalyzes the first mannose trimming step associated with processing of high mannose oligosaccharide structures by removing a terminal mannose sugar from the oligosaccharide. N-terminal glycosylation involves the addition and removal of various monosaccharide sugars in both the endoplasmic reticulum (ER) and Golgi compartments (Komfeld et al., Ann. Rev. Biochem. 54: 631-664, 1985). In the ER, the N-linked glycosylation is accompanied by the folding of nascent glycoproteins into their native structure through interactions with molecular chaperones (Ellgaard et al., Science 286: 1882-1888, 1999; Jakob et al., J. Cell Biol. 142: 1223-1233, 1998). This process has been termed ER quality control, and if the process is blocked due to a misfolded protein, the onset of ER associated degradation, or ERAD, of the protein typically occurs (Ellgaard et al., Curr. Opin. Cell Biol. 13: 431-437, 2001; Sifers, Science 299: 1330-1331, 2003; Oda et al., Science 299: 1394-1397, 2003; Molinari et al., Science 299:1397-1400, 2003; Hurtley et al., Ann. Rev. Cell Biol. 5: 277-307, 1989). The removal of a terminal mannose sugar from Man9 to Man8 by the alpha 1,2 mannosidase I enzyme (MAN1C1) has been shown to affect the onset of the ERAD response (Liu et al., J. Biol. Chem. 274: 5861-5867, 1999; Grinna et al. J. Biol. Chem. 255, 2255-2258, 1980).
In view of the toxicity of protein production inducers such as sodium butyrate, there exists a need for other means of increasing overall production of recombinant proteins in cell culture.